The long term objectives of this research proposal are to understand at the molecular level the basic mechanisms controlling transcription and replication of vesicular stomatitis virus (VSV) and its defective interfering (DI) particle RNAs. VSV, the prototypical rhabdovirus with a single-stranded negative-sense RNA genome is an excellent model system for numerous other disease-causing negative-strand RNA viruses. It has also been used as a model virus of choice for studying basic mechanisms of negative-strand RNA virus genome transcription and replication and virus assembly. The proposed research is designed to gain knowledge on various transcription and replication regulating sequences and their functions. A recently developed reverse genetic system that utilizes only cDNAs for VSV proteins and RNA will be used to introduce specific alterations into the genome of VSV. The effect of these mutations on transcription and replication of VSV RNA will be measured by various biochemical means. identification and characterization of the regulatory sequences by the methods described above will be essential to gain insight into the mechanisms of RNA transcription and replication in this group of important human pathogens. The specific aims of this proposal are as follows: 1. Competition and interference studies with different size DI particles. 2. Identification and characterization of signals for encapsidation and replication of DI particle genomic RNA. 3. Identification and characterization of various transcription regulatory sequence elements within the genome of VSV. 4. Evaluation of the role of sequences at or near the leader-N gene junction of VSV in transcription attenuation or readthrough.